Gram-negative septic shock, which is characterized by a high mortality rate and is responsible for thousands of deaths annually, appears to result from a cascade of events triggered by the action of bacterial endotoxin.
Endotoxin is a lipopolysaccharide (LPS) which is a major constituent of the outer-leaflet of the membranes of Gram-negative bacteria. Structural studies have shown that it consists of the following three distinct domains: 1) the antigen region, which is a strain-specific polysaccharide moiety and determines the antigenic specificity of the organism; 2) the core region, which is relatively conserved with respect to its sugar composition and may play a role in maintaining the integrity of the outer membrane; and 3) the lipid A region, which is also conserved and functions as a hydrophobic anchor holding lipopolysaccharide in place. The lipid A portion of lipopolysaccharide constitutes most of the outer monolayer of the outer membrane in Gram-negatives.
Lipopolysaccharide is known to trigger many pathophysiological events in mammals, either when it is injected or when it accumulates due to Gram-negative infection. In general, the hydrophobic lipid A moiety is responsible for these pathophysiological effects, which tend to be either immunostimulatory or toxic. In the former category there are events such as B-lymphocyte mitogenesis, macrophage activation, and the induction of tumor necrosis in certain experimental systems. In the latter (toxic) category there are responses such as peripheral vascular collapse ("endotoxic" or septic shock), pulmonary hypertension, pulmonary edema, disseminated intravascular coagulopathy and pyrogenicity.
Of particular importance concerning the lethal effects of LPS is the observation that nanogram quantities of LPS can induce the release of mediators such as tumor necrosis factor-.alpha., (TNF-.alpha.) interleukin-1 (IL-1), and interleukin-6 (IL-6) (1-5). The release of mediators such as tumor necrosis factor-a (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6) is thought to produce the toxicity associated with endotoxemia. However, despite the high mortality rate of endotoxic shock, relatively little is known about the biochemical mechanisms involved in LPS-induced events, e.g., TNF and IL-1 release, although an amplification system is suggested given that nanogram quantities of LPS can produce severe toxicity in animals. Most amplification pathways involve receptors and various enzyme cascades, thus allowing for several points of antagonism within the system. Although certain steps in LPS action are known, such as the stimulation of phosphoinositide hydrolysis, a transient increase in intracellular Ca.sup.++ levels, and the activation of protein kinase C and phospholipase A.sub.2, the lack of specific information on the cellular mechanisms involved in LPS action has impeded the development of therapeutic agents for preventing endotoxin shock.
Macrophages are particularly important cells in LPS-mediated TNF-.alpha., IL-1, and IL-6 release. Although a detailed understanding of the cellular and biochemical processes through which LPS activates macrophages is unknown several lines of indirect evidence suggest that G-proteins might be involved in LPS action, which would be consistent with a receptor-linked cellular amplification pathway. Our finding that LPS stimulates a macrophage membrane-associated GTPase, an activity that is a hallmark of G-protein involvement, further supports a role for G-proteins in endotoxicity.
In our earlier application, we disclosed an assay for identifying compounds that inhibit the release of endotoxic shock mediators, such as TNF, by measuring the compounds ability to inhibit LPS-induced GTPase in an in vitro assay.
The assay basically comprises testing candidate compounds to determine if they are effective in blocking LPS-induced GTPase activity in macrophage membranes in vitro. Because previous studies suggested that a G-protein (a GTP-binding protein that becomes active when compounds bind to receptor and cause the G-protein to activate other cellular effector molecules) may participate in LPS action, we evaluated the effects of LPS on GTPase activity in membranes isolated from a macrophage (RAW 264.7) cell line. (G-proteins cause hydrolysis of GTP, hence, they are GTPases.) We found that LPS induced substantial GTPase activation (200-300% above basal), and kinetic analyses indicated that the maximal LPS-stimulated increase in velocity is observed within 15 min, that it is a low K.sub.m (GTP) activity, that it can be enhanced by ammonium sulfate, and that it appears to be pertussis toxin-insensitive.
Moreover, we found the LPS-enhanced GTPase activity was not antagonized by phosphatase/ATPase inhibitors such as p-nitrophenyl-phosphate, ouabain, bafilomycin or N-ethylmaleimide, and in fact was potentiated by the addition of ATP or ADP. We found a synergistic effect on GTPase activity when both LPS and ATP or ADP are added to the macrophage assay. These data suggest that LPS may modulate the coupling between GTPase and a purinoreceptor, which is a class of cell surface hormone receptors that can bind ATP, ADP or related adenine nucleotides, and can serve to regulate cellular function via effects on selected G-proteins.
We also found that the LPS precursor, lipid X, which can reduce the lethal effects of LPS endotoxin, caused a dose-dependent inhibition of the LPS-mediated stimulation of GTPase activity. Half-maximal inhibition was seen at the same ratio of lipid X to LPS known to be effective in preventing endotoxin effects in vivo, i.e. at a one to one weight ratio. These effects are specific because other phospholipids, detergents and glycosides neither stimulated basal GTPase activity nor inhibited LPS-induced GTPase activity. Also, other studies showed that GTPase activity was not due to ATPase or to guanylate cycle activities.
In our earlier application we also disclosed our discovery that 2-methylthio-ATP blocks both LPS-induced GTPase activity and TNF production in RAW 2647 macrophages which provides additional evidence that GTPase is a determinative pathway for mediating endotoxicity.
Obviously, it would be advantageous to have compounds that could be useful as therapeutic agents in methods to prevent endotoxin shock. It also would be useful to have compounds which inhibit the release of tumor necrosis factor (TNF) and interleukin-1 (IL-1).